Method and reagents for the enhancement of virus transduction in the bladder epithelium

ABSTRACT

Agents and methods for enhancing recombinant virus transduction in the bladder epithelium are described. A first method involves contacting the luminal surface of the bladder with a composition comprising a transduction enhancing agent and an oncolytic virus. Alternatively, the luminal surface of the bladder can be contacted first with a pretreatment composition comprising a transduction enhancing agent and, subsequently, with a composition comprising an oncolytic virus. Bladder treatment compositions comprising a transduction enhancing agent and an oncolytic virus are also described.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/327,869, filed Dec. 26, 2002, which application isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the treatment of bladdercancer with viral therapy agents and, in particular, to agents andmethods for enhancing recombinant oncolytic virus transduction of thebladder epithelium.

2. Background of the Technology

Bladder cancer is a commonly occurring cancer and more than 50,000 newcases are diagnosed every year. Bladder cancer is a superficial diseaseconfined to the mucosa in the majority of patients. Of the varioustherapeutic modalities available, transurethral resectioning of thetumor is considered to be the most effective treatment for themanagement of superficial bladder cancer. However, 70% of thesesuperficial bladder tumors will recur after endoscopic resectioning, and20% progress to life-threatening invasive diseases within 2 years ofcystectomy. See Raghavan, et al., “Biology and Management of BladderCancer”, N. Engl. J. Med., 322, 16, 1129-1138 (1990).

Gene therapy has also been used for the treatment of bladder cancer.See, for example, Brewster, et al., Eur. Urol. 25, 177-182 (1984);Takahashi, et al., Proc. Natl. Acad. Sci. USA 88, 5257-5261 (1991); andRosenberg, J. Clin. Oncol., 10, 180-199 (1992).

In vitro studies using cell lines derived from human bladder tissueshave demonstrated efficient transgene expression following infectionwith recombinant adenovirus. Bass, et al., Cancer Gene Therapy 2, 2,97-104 (1995). Experiments in vivo have also shown adenovirus transgeneexpression in the urinary bladder of rodents after intravesicaladministration. Bass, et al., supra; Morris. et al., J. Urology, 152,506-550 (1994). In vitro experiments with wild-type adenovirusdemonstrate that virus attachment and internalization is not influencedby benzyl alcohol, but do demonstrate an enhanced uncoating of thevirion. Blixt. et al., Arch. Virol., 129, 265-277 (1993).

In vivo studies have demonstrated that various agents (e.g. acetone,DMSO, protamine sulfate) can break down the protective “mucin” layerthat protects the bladder epithelium from bacteria, viruses and otherpathogens. See, for example, Monson, et al., J. Urol., 145, 842-845(1992) and Parsons, et al., J. Urol., 143, 139-142 (1990). Methods ofmodifying the bladder surface to enhance gene transfer have also beendisclosed. Siemens. et al., “Evaluation of Gene Transfer Efficiency byViral Vectors to Murine Bladder Epithelium”, J. of Urology, 165, 667-671(2001).

U.S. Pat. No. 6,165,779 discloses a gene delivery system formulated in abuffer comprising a delivery-enhancing agent such as ethanol or adetergent. The gene delivery system may be a recombinant viral vectorsuch as an adenoviral vector.

There still exists a need, however, for improved gene therapy methodsand agents which can accomplish direct, optimal, in vivo gene deliveryto the bladder epithelium.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, a method for treatingcancer of the bladder is provided. According to this aspect of theinvention, the method involves: contacting the luminal surface of thebladder with a pretreatment composition comprising a transductionenhancing agent; and subsequently contacting the luminal surface of thebladder with a composition comprising an oncolytic virus; wherein thetransduction enhancing agent is a mono-, di-, or poly-saccharide havinga lipophilic substituent. The transduction enhancing agent can have thefollowing general formula (I) or the following general formula (II):

wherein X is a sulfur or oxygen atom, R¹ is an alkyl group and each R²is independently hydrogen or a moiety represented by:

wherein R¹ is an alkyl group. The pretreatment composition can furtherinclude an oxidizing agent. The oncolytic virus can be an oncolyticadenovirus such as CG8840. The oncolytic virus composition can furtherinclude a chemotherapeutic agent such as docetaxel.

According to a second aspect of the invention, a method for treatingcancer of the bladder is provided. According to this aspect of theinvention, the method includes contacting the luminal surface of thebladder with a pretreatment composition comprising about 0.01 to about0.2% by weight sodium oxychlorosene and, subsequently, contacting theluminal surface of the bladder with a composition comprising anoncolytic virus.

According to a third aspect of the invention, a method of treatingcancer of the bladder is provided. According to this aspect of theinvention, the method includes: contacting the luminal surface of thebladder with a pretreatment composition comprising a transductionenhancing agent having a structure represented by the chemical formula:

wherein x and y are positive integers; and subsequently contacting theluminal surface of the bladder with a composition comprising anoncolytic virus. According to a preferred embodiment of the invention, xis 6 and y is 8-10 and the pretreatment composition comprises about 0.02to about 0.05 wt. % of the transduction enhancing agent.

According to a fourth aspect of the invention, a method of treatingcancer of the bladder is provided. According to this aspect of theinvention, the method includes: contacting the luminal surface of thebladder with a pretreatment composition comprising a transductionenhancing agent having a structure represented by the following generalformula (I) or the following general formula (II):

wherein x is a positive integer and subsequently contacting the luminalsurface of the bladder with a composition comprising an oncolytic virus.

According to a fifth aspect of the invention, a composition comprising atransduction enhancing agent and an oncolytic virus is provided.According to this aspect of the invention, the transduction enhancingagent is a mono-, di-, or poly-saccharide having a lipophilicsubstituent. For example, the transduction enhancing agent can be acompound having the following general formula (I) or the followinggeneral formula (II):

wherein X is a sulfur or oxygen atom, R¹ is an alkyl group and each R²is independently hydrogen or a moiety represented by:

wherein R¹ is an alkyl group. The oncolytic virus can be an oncolyticadenovirus such as CG8840. The oncolytic virus composition can furtherinclude a chemotherapeutic agent such as docetaxel. A method fortreating cancer of the bladder comprising contacting the luminal surfaceof the bladder with a composition as set forth above is also provided.

According to a sixth aspect of the invention, a composition comprisingsodium oxychlorosene and an oncolytic virus is provided. The oncolyticvirus can be an oncolytic adenovirus such as CG8840. The oncolytic viruscomposition can further include a chemotherapeutic agent such asdocetaxel. A method for treating cancer of the bladder comprisingcontacting the luminal surface of the bladder with a composition as setforth above is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be better understood with reference to theaccompanying drawings in which:

FIGS. 1A and 1B are photographs showing a murine bladder afterpretreatment with a 15% ethanol solution followed by infection withAd-LacZ wherein FIG. 1A shows the outside surface of the bladder andFIG. 1B shows the luminal bladder surface;

FIGS. 1C and 1D are photographs showing a murine bladder afterpretreatment with a 20% ethanol solution followed by infection withAd-LacZ wherein FIG. 1C shows the outside surface of the bladder andFIG. 1D shows the luminal bladder surface;

FIGS. 1E and 1F are photographs showing a murine bladder afterpretreatment with a 25% ethanol solution followed by infection withAd-LacZ wherein FIG. 1E shows the outside surface of the bladder andFIG. 1F shows the luminal bladder surface;

FIGS. 1G and 1H are photographs showing a murine bladder afterpretreatment with a 30% ethanol solution followed by infection withAd-LacZ wherein FIG. 1G shows the outside surface of the bladder andFIG. 1H shows the luminal bladder surface;

FIG. 2A ia a photograph showing a cross section of a murine bladdercontrol;

FIGS. 2B and 2C are photographs showing the cross section of a murinebladder after pretreatment with a 30% ethanol solution followed byinfection with Ad-LacZ;

FIGS. 3A-3F are photographs showing the cross section of a murinebladder after pretreatment with a 25% ethanol solution followed byinfection with Ad-LacZ wherein FIGS. 3A, 3C and 3E were taken at 40× andFIGS. 3B, 3D and 3F were taken at 100× magnification;

FIGS. 4A-4F are photographs showing the cross section of a murinebladder after pretreatment with a 30% ethanol solution followed byinfection with Ad-LacZ wherein FIGS. 4A, 4C and 4E were taken at 40× andFIGS. 4B, 4D and 4F were taken at 100× magnification;

FIGS. 5A-5D are photographs showing two murine bladders afterpretreatment with a 4% poloxomer 407 solution followed by infection withAd-LacZ wherein FIGS. 5A and 5B show the outside and luminal surfaces,respectively, of the first bladder and FIGS. 5C and 5D show the outsideand luminal surfaces, respectively, of the second bladder;

FIGS. 6A-6D are photographs showing two murine bladders after infectionwith a composition comprising lipofectamine and Ad-LacZ wherein FIGS. 6Aand 6B show the outside and luminal surfaces, respectively, of the firstbladder and FIGS. 6C and 6D show the outside and luminal surfaces,respectively, of the second bladder;

FIGS. 7A-7D are photographs showing two murine bladders after infectionwith a composition comprising In vivo geneSHUTTLE™ and Ad-LacZ whereinFIGS. 7A and 7B show the outside and luminal surfaces, respectively, ofthe first bladder and FIGS. 7C and 7D show the outside and luminalsurfaces, respectively, of the second bladder;

FIGS. 8A-8N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 5 minutes followedby infection with Ad-LacZ wherein FIGS. 8A and 8B show the outside andluminal surfaces, respectively, of the first bladder, FIGS. 8C and 8Dshow the outside and luminal surfaces, respectively, of the secondbladder, FIGS. 8E and 8F show the outside and luminal surfaces,respectively, of the third bladder, FIGS. 8G and 8H show the outside andluminal surfaces, respectively, of the fourth bladder, FIGS. 8I and 8Jshow the outside and luminal surfaces, respectively, of the fifthbladder, FIGS. 8K and 8L show the outside and luminal surfaces,respectively, of the sixth bladder, and FIGS. 8M and 8N show the outsideand luminal surfaces, respectively, of the seventh bladder;

FIGS. 9A-9N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 15 minutes followedby infection with Ad-LacZ wherein FIGS. 9A and 9B show the outside andluminal surfaces, respectively, of the first bladder, FIGS. 9C and 9Dshow the outside and luminal surfaces, respectively, of the secondbladder, FIGS. 9E and 9F show the outside and luminal surfaces,respectively, of the third bladder, FIGS. 9G and 9H show the outside andluminal surfaces, respectively, of the fourth bladder, FIGS. 9I and 9Jshow the outside and luminal surfaces, respectively, of the fifthbladder, FIGS. 9K and 9L show the outside and luminal surfaces,respectively, of the sixth bladder, and FIGS. 9M and 9N show the outsideand luminal surfaces, respectively, of the seventh bladder;

FIGS. 10A and 10B are photographs showing the cross section of themurine bladders of FIGS. 8C and 8I, respectively;

FIGS. 11A and 11B are photographs showing the cross section of themurine bladders of FIGS. 9C and 9I, respectively;

FIGS. 12A-12F are photographs showing the cross section of a murinebladder after pretreatment with a 0.2% oxychlorosene solution for 5minutes followed by infection with Ad-LacZ wherein FIGS. 12A, 12C and12E were taken at 40× and FIGS. 12B, 12D and 12F were taken at 100×magnification;

FIGS. 13A-13F are photographs showing the cross section of a murinebladder after pretreatment with a 0.2% oxychlorosene solution for 15minutes followed by infection with Ad-LacZ wherein FIGS. 13A, 13C and13E were taken at 40× and FIGS. 13B, 13D and 13F were taken at 100×magnification;

FIG. 14A is a photograph showing the luminal surface of a murine bladderafter pretreatment with a 0.1% oxychlorosene solution followed byinfection with Ad-LacZ;

FIGS. 14B and 14C are photographs showing the cross section of themurine bladder of FIG. 14A wherein FIG. 14B was taken at 40× and FIG.14C was taken at 100× magnification;

FIG. 15A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.2% oxychlorosene solution followedby infection with Ad-LacZ;

FIGS. 15B and 15C are photographs showing the cross section of themurine bladder of FIG. 15A wherein FIG. 15B was taken at 40× and FIG.15C was taken at 100× magnification;

FIG. 16A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.2% oxychlorosene solution followedby infection with Ad-LacZ;

FIGS. 16B and 16C are photographs showing the cross section of themurine bladder of FIG. 16A wherein FIG. 16B was taken at 40× and FIG.16C was taken at 100× magnification;

FIG. 17A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.4% oxychlorosene solution followedby infection with Ad-LacZ;

FIGS. 17B and 17C are photographs showing the cross section of themurine bladder of FIG. 17A wherein FIG. 17B was taken at 40× and FIG.17C was taken at 100× magnification;

FIG. 18A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.4% oxychlorosene solution followedby infection with Ad-LacZ;

FIGS. 18B and 18C are photographs showing the cross section of themurine bladder of FIG. 18A wherein FIG. 18B was taken at 40× and FIG.18C was taken at 100× magnification;

FIG. 19A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.02% polidocanol solution followed byinfection with Ad-LacZ;

FIGS. 19B and 19C are photographs showing the cross section of themurine bladder of FIG. 19A wherein FIG. 19B was taken at 40× and FIG.19C was taken at 100× magnification;

FIG. 20A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.02% polidocanol solution followed byinfection with Ad-LacZ;

FIGS. 20B and 20C are photographs showing the cross section of themurine bladder of FIG. 20A wherein FIG. 20B was taken at 40× and FIG.20C was taken at 100× magnification;

FIG. 21A and 21B are photographs showing the outside and luminalsurfaces, respectively, of a first murine bladder after pretreatmentwith a 0.05% polidocanol solution followed by infection with Ad-LacZ;

FIGS. 21C and 21D are photographs showing the cross section of themurine bladder of FIG. 21A wherein FIG. 21B was taken at 40× and FIG.21C was taken at 100× magnification;

FIG. 22A and 22B are photographs showing the outside and luminalsurfaces, respectively, of a second murine bladder after pretreatmentwith a 0.05% polidocanol solution followed by infection with Ad-LacZ;

FIGS. 22C and 22D are photographs showing the cross section of themurine bladder of FIG. 22A wherein FIG. 22B was taken at 40× and FIG.22C was taken at 100× magnification;

FIG. 23A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.2% polidocanol solution followed byinfection with Ad-LacZ;

FIGS. 23B and 23C are photographs showing the cross section of themurine bladder of FIG. 23A wherein FIG. 23B was taken at 40× and FIG.23C was taken at 100× magnification;

FIG. 24A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.2% polidocanol solution followed byinfection with Ad-LacZ;

FIGS. 24B and 24C are photographs showing the cross section of themurine bladder of FIG. 24A wherein FIG. 24B was taken at 40× and FIG.24C was taken at 100× magnification;

FIG. 25A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.02% n-dodecyl β-D-maltoside solutionfollowed by infection with Ad-LacZ;

FIGS. 25B and 25C are photographs showing the cross section of themurine bladder of FIG. 25A wherein FIG. 25B was taken at 40× and FIG.25C was taken at 100× magnification;

FIG. 26A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.02% n-dodecyl β-D-maltoside solutionfollowed by infection with Ad-LacZ;

FIGS. 26B and 26C are photographs showing the cross section of themurine bladder of FIG. 26A wherein FIG. 26B was taken at 40× and FIG.26C was taken at 100× magnification;

FIG. 27A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.05% n-dodecyl β-D-maltoside solutionfollowed by infection with Ad-LacZ;

FIGS. 27B and 27C are photographs showing the cross section of themurine bladder of FIG. 27A wherein FIG. 27B was taken at 40× and FIG.27C was taken at 100× magnification;

FIG. 28A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.05% n-dodecyl β-D-maltoside solutionfollowed by infection with Ad-LacZ;

FIGS. 28B and 28C are photographs showing the cross section of themurine bladder of FIG. 28A wherein FIG. 28B was taken at 40× and FIG.28C was taken at 100× magnification;

FIG. 29A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.2% n-dodecyl β-D-maltoside solutionfollowed by infection with Ad-LacZ;

FIGS. 29B and 29C are photographs showing the cross section of themurine bladder of FIG. 29A wherein FIG. 29B was taken at 40× and FIG.29C was taken at 100× magnification;

FIG. 30A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.2% sodium salt of dedecylbenzenesulfonic acid solution followed by infection with Ad-LacZ;

FIGS. 30B and 30C are photographs showing the cross section of themurine bladder of FIG. 30A wherein FIG. 30B was taken at 40× and FIG.30C was taken at 100× magnification;

FIGS. 31A-31E are photographs showing the luminal surfaces of murinebladders treated with alkyl maltoside and alkyl maltopyranosidepretreating agents having various length alkyl side chains; and

FIGS. 32A-32C are photographs showing the luminal surfaces of murinebladders treated with sodium alkyl sulfate pretreating agents havingvarious length alkyl chains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the use of transduction enhancingagents to render the bladder umbrella cell layer more susceptible toinfection with a viral gene delivery vehicle than it would be withouttreatment. Exemplary transduction enhancing agents according to theinvention include: dodecyl surfactants; dodecylmaltosides; dodecylalcohol polyoxyethylene ethers (i.e., polidocanol); and sodiumdodecylbenzenesulphonic acid/hypochlorous acid complex (i.e.,oxychlorosene).

According to the invention, the luminal surface of the bladder can betreated with a composition comprising a transduction enhancing agentprior to infection with a viral gene delivery vehicle. The viral genedelivery vehicle can be an oncolytic virus used to treat bladder cancer.Oncolytic viruses for use in practicing the invention include, but arenot limited to, adenovirus, herpes simplex virus (HSV), reovirus,vesicular stomatitis virus (VSV), newcastle disease virus, vaciniavirus, influenza virus, West Nile virus, coxsackie virus, poliovirus andmeasles virus. Of particular interest in practicing the invention areoncolytic viruses that exhibit preferential expression in particulartissue types (i.e., in the bladder urothelium). An oncolytic adenovirusof this type is disclosed, for example, in Zhang. et al.,“Identification of Human Uroplakin II Promoter and Its Use in theConstruction of CG8840, a Urothelium-specific Adenovirus Variant thatEliminates Established Bladder Tumors in Combination with Docetaxel”,Cancer Research, 62, 3743-3750 (2002) and in co-owned U.S. patentapplication Ser. No. 09/814,292, which is expressly incorporated byreference herein. Chemotherapeutic agents for use in combination therapywith oncolytic viruses are described, for example, in co-owned U.S.patent application Ser. No. 09/814,357, which is expressly incorporatedby reference herein.

Alternatively, the viral gene delivery vehicle can be any gene therapydelivery vehicle known in the art for use in gene therapy, including,but not limited to, an adenovirus, an adeno-associated virus (AAV), alentivirus, a retrovirus, a herpes virus, etc. Exemplary gene therapyadenoviral agents are disclosed in U.S. Pat. No. 6,165,779. The presentinventors have found that pre-treating mouse bladders with aqueoussolutions of various compounds consistently increased transduction togreater than 60% of the bladder surface, versus an untreated percenttransduction of no more than 10%.

In addition to pre-treatment of the bladder surface with thetransduction enhancing agent, the present invention includesco-administration of the viral gene delivery vehicle and thetransduction enhancing agent to the bladder and to co-formulations ofany one of the transduction enhancing agents with a recombinant viralgene delivery vehicle.

Composition and Chemistry of Reagents Used to Enhance AdenovirusTransduction in the Bladder Epithelium

Several classes of compounds, surfactants, and pre-made reagents weretested in order to find those which increased gene transfer ortransduction by a viral gene delivery vehicle in the bladder. Anoncolytic adenovirus, CG884, was used as an exemplary viral gene therapyvehicle. The reagents evaluated can be classified by their physical orchemical properties and structure.

First, the reagents can be grouped as a single compound or as a mixedreagent (i.e., a mixture of compounds). Single compounds evaluatedinclude non-ionic surfactants, alcohols, polymers and ionic surfactants.The ionic surfactants evaluated included: 4% Poloxamer 407 (Pluronic®127); 4% poloxamer 188 (Pluronic® F68); 0.02%-0.5% Polidocanol; 0.1%n-dodecyl-b-D-glucopyranoside (which can also be classified as asugar-based surfactant); 0.02-0.5% n-dodecyl-b-D-maltoside (which canalso be classified as a sugar-based surfactant); 0.1% Tween® 20; 0.1%Triton® X-100; 0.1% Forlan® C-24 (PEG Cholesterol); 0.1%decyl-b-D-maltoside (which can also be classified as a sugar-basedsurfactant); 0.1% 6-cyclohexylhexyl-β-D-maltoside (which can also beclassified as a sugar-based surfactant); and 0.1% Tromboject® (sodiumtetradecyl sulfate).

Alcohols evaluated include 0.1%-3% benzyl alcohol and 10%-30% ethanol.Polymers evaluated include 0.4% HPMC 2910; 0.4% PVA; 0.4% PVP; and 100mg/ml Poly-Lysine. Ionic surfactants evaluated include: 0.1% DC-Chol[Cholesteryl 3b-N-(dimethylaminoethyl) carbamate]; 0.2% sodium salt ofDodecyl benzenesulfonic acid; and 0.1% sodium dodecyl sulfate. Mixedreagents evaluated include: In vivo GeneSHUTTLE™ (a reagent comprisingDOTAP+Cholesterol available from Qbiogene of Carlsbad, Calif.) and0.1%-0.4% Oxychlorosene (sodium dodecylbenzenesulphonicacid/hypochlorous acid complex).

Effect of Ethanol Pretreatment on Adenovirus-Mediated Gene Transfer andExpression in the Bladder Epithelium of Rodents

A study was conducted to evaluate the effect of ethanol pretreatment onadenovirus-mediated gene transfer and expression in the bladderepithelium of rodents.

Test Materials

Ad-βgal virus was made as a frozen formulation using standard conditionsknown in the art for freezing and formulation of adenovirus. The vehiclefor the virus arm was PBS plus 10% glycerol. Pretreatment agents were5%, 10%, 15%, 20% and 30% GLP grade ethanol, respectively, in PBS-10%glycerol solution.

Animals

80 female BALB/c mice were used for this study. Female animals arechosen because of the ease of urethral cannulation and vesicleinstillation. The mice were approximately 10 to 12 weeks on the day ofthe start of the experiment.

Treatment Regimen

Animals were assigned to each group as shown in the following table.

TABLE 1 Effect of Ethanol pretreatment Animals Virus dose Group per TestEthanol (particles/ No. group Article Route pretreatment animal) DoseRegimen 1 8-10 Vehicle Intravesical — — 100 ml PBS- 10% glycerol on Day1 2 8-10 Ad-βgal Intravesical — 1.3 × 10¹¹ Ad-βgal on Day 1 3 8-10Ad-βgal Intravesical 5% ethanol 1.3 × 10¹¹  5% ethanol pretreatmentfollowed by virus administration on Day 1 4 8-10 Ad-βgal Intravesical10% ethanol 1.3 × 10¹¹ 10% ethanol pretreatment followed by virusadministration on Day 1 5 8-10 Ad-βgal Intravesical 15% ethanol 1.3 ×10¹¹ 15% ethanol pretreatment followed by virus administration on Day 16 8-10 Ad-βgal Intravesical 20% ethanol 1.3 × 10¹¹ 20% ethanolpretreatment followed by virus administration on Day 1 7 8-10 Ad-βgalIntravesical 25% ethanol 1.3 × 10¹¹ 25% ethanol pretreatment followed byvirus administration on Day 1 8 8-10 Ad-βgal Intravesical 30% ethanol1.3 × 10¹¹ 30% ethanol pretreatment followed by virus administration onDay 1For the data in Table 1, the concentration of Ad-βgal virus was 1.3×10¹²vp/mi as determined by optical density measurements.

Treatment Procedure

1. Animals were anesthetized with isoflurane and a 24 g catheterintroduced through the urethra into the bladder.

2. Residual urine was emptied and the bladder was flushed 3 times with100-150 μl each of PBS.

3. In test animals, bladders were pretreated for 20 minutes with 0.1 mlof 5, 10, 15, 20, 25 or 30% ethanol solution, respectively, and thenrinsed 3 times with 100-150 μl of PBS.

4. Ad-βgal viruses diluted in 0.1 ml of PBS-10% glycerol wereadministered intravesically into the bladder and retained in the bladderfor 45 minutes. A knot was placed around the urethral orifice to preventleakage of the virus and to prevent the catheter from dislodging.

5. Treatment was stopped by withdrawing the virus and flushing thebladders 3 times with 100-150 μl of PBS. If the catheter became clogged,the washing step was avoided so that the virus was flushed out in theurine. However, the use of this procedure may prevent determination ofthe viral resident time in the bladder.

Measurement/Determinations

The clinical condition of the animals was observed before dosing on theday of treatment and the animals were observed daily during theexperimental period.

Assessment of β-Galactosidase Activity

Animals were killed 48 hours after treatment. Bladders were filled with0.1 ml whole organ fixative: 2% Neutral buffered formalin, 2%glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4. Bladders were thenremoved and immersed in whole organ fixative for 1 hr. Thereafter, thebladders were cut open longitudinally, rinsed (2 mM MgCl₂, 0.1%deoxycholate, 0.2% Triton) for 24 hours at 4° C., and submerged intoX-gal staining solution. Transgene expression in the luminal epitheliumof the longitudinally opened bladders was empirically determined.

Histopathology

Bladders fixed in whole organ fixative were sectioned and stained withhematoxylin-eosin for histologic examination.

Results

Pretreatment of the luminal bladder surface with various concentrationsof ethanol (i.e., 15%, 20%, 25%, and 30 wt. %) for 20 minutes resultedin 10-20% transduction. FIGS. 1-4 show transduction of murine bladdersafter pretreatment with ethanol. FIGS. 1A and 1B are photographs showinga murine bladder after pretreatment with a 15% ethanol solution followedby infection with Ad-LacZ. FIG. 1A shows the outside surface of thebladder and FIG. 1B shows the luminal bladder surface. FIGS. 1C and 1Dare photographs showing a murine bladder after pretreatment with a 20%ethanol solution followed by infection with Ad-LacZ. FIG. 1C shows theoutside surface of the bladder and FIG. 1D shows the luminal bladdersurface. FIGS. 1E and 1F are photographs showing a murine bladder afterpretreatment with a 25% ethanol solution followed by infection withAd-LacZ. FIG. 1E shows the outside surface of the bladder and FIG. 1Fshows the luminal bladder surface. FIGS. 1G and 1H are photographsshowing a murine bladder after pretreatment with a 30% ethanol solutionfollowed by infection with Ad-LacZ. FIG. 1G shows the outside surface ofthe bladder and FIG. 1H shows the luminal bladder surface. As can beseen from FIG. 1, higher concentrations of ethanol resulted in greaterlevels of transduction as measured by staining.

FIG. 2A is a photograph showing a cross section of a murine bladdercontrol (i.e., no pretreatment). FIGS. 2B and 2C are photographs showingthe cross section of a murine bladder after pretreatment with a 30%ethanol solution followed by infection with Ad-LacZ.

FIGS. 3A-3F are photographs showing the cross section of three murinebladders after pretreatment with a 25% ethanol solution followed byinfection with Ad-LacZ. FIGS. 3A and 3B are photographs showing thecross-section of the first murine bladder, FIGS. 3C and 3D arephotographs showing the cross-section of the second murine bladder, andFIGS. 3E and 3F are photographs showing the cross-section of the thirdmurine bladder. FIGS. 3A, 3C and 3E were taken at 40× and FIGS. 3B, 3Dand 3F were taken at 100× magnification.

FIGS. 4A-4F are photographs showing the cross section of three murinebladders after pretreatment with a 30% ethanol solution followed byinfection with Ad-LacZ. FIGS. 4A and 4B are photographs showing thecross-section of the first murine bladder, FIGS. 4C and 4D arephotographs showing the cross-section of the second murine bladder, andFIGS. 4E and 4F are photographs showing the cross-section of the thirdmurine bladder. FIGS. 4A, 4C and 4E were taken at 40× and FIGS. 4B, 4Dand 4F were taken at 100× magnification.

Effect of Chemical Agent Pretreatment on Adenovirus-Mediated GeneTransfer and Expression in the Bladder Epithelium of Rodent

A study was conducted to evaluate the effect of chemical agentpretreatment on adenovirus-mediated gene transfer and expression in thebladder epithelium of rodents.

Test Materials

Ad-βgal virus was made at CGI, as a frozen formulation using standardconditions known in the art for freezing and formulation of adenovirus.The vehicle for the virus arm was PBS plus 10% glycerol.

Animals

152 female BALB/c mice were used this study. Female animals were chosenbecause of the ease of urethral cannulation and vesicle instillation.The mice were approximately 10 to 12 weeks on the day of the start ofthe experiment.

Treatment Regimen

Animals were assigned to each group shown in the following table. Theroute of administration of the chemical agent and virus wasintravesical.

TABLE 2 Effect of Chemical Agent Pretreatment Animals Virus dose Groupper Test (particles/ No. group Article Chemical agent animal) DoseRegimen 1 2 Vehicle 4% Poloxamer 407 — 100 ml of 4% (Pluronic 127)Poloxamer 407 (Pluronic 127) in PBS- 10% glycerol on Day 1 2 6-8 Ad-βgal4% Poloxamer 407 1.3 × 10¹¹   4% Poloxomer 407 (Pluronic 127) (Pluronic127) pretreatment followed by virus administration on Day 1 3 2 Vehicle4% Poloxamer 188 100 ml of 4% (Pluronic F68) Poloxamer 188 (PlluronicF68) in PBS-10% glycerol on Day 1 4 6-8 Ad-βgal 4% Poloxamer 188 1.3 ×10¹¹   4% Poloxomer 188 (Pluronic F68) (Pluronic F68) pretreatmentfollowing by virus administration on Day 1 5 2 Vehicle Lipofectamine 100ml of 2000 Lipofectamine 2000 (20 mg/ml) in PBS 10% glycerol. 6 6-8Ad-βgal Lipofectamine 0.65 × 10¹¹   1.25 mg of 2000 Lipofectamine 2000mixed with virus administration on Day 1 7 2 Vehicle 3% Benzyl 100 μl of3% Benzyl Alcohol Alcohol in PBS-10% glycerol on Day 1 8 6-8 Ad-βgal 3%Benzyl 1 × 10¹¹ 3% Benzyl Alcohol Alcohol pretreatment followed by virusadministration on Day 1 9 2 Vehicle 0.2% 0.2% OxychloroseneOxychlorosene in PBS-10% glycerol on Day 1 10 6-8 Ad-βgal 0.2% 1.3 ×10¹¹   0.2% Oxychlorosene Oxychlorosene pretreatment (only wash)followed by virus administration on Day 1 11 6-8 Ad-βgal 0.2% 1.3 ×10¹¹   0.2% Oxychlorosene Oxychlorosene pretreatment (5 min) followed byvirus administration on Day 1 12 6-8 Ad-βgal 0.2% 1.3 × 10¹¹   0.2%Oxychlorosene Oxychlorosene pretreatment (15 min) followed by virusadministration on Day 1 13 2 Vehicle 0.05% Polidocanol 0.05% Polidocanolin PBS-10% glycerol on Day 1 14 6-8 Ad-βgal 0.05% Polidocanol 1.3 ×10¹¹   0.05% Polidocanol pretreatment followed by virus administrationon Day 1 15 2 Vehicle 0.1% DC-Chol 0.1% DC-Chol in PBS-10% glycerol onDay 1 16 6-8 Ad-βgal 0.1% DC-Chol 1.3 × 10¹¹   0.1% DC-Chol pretreatmentfollowed by virus administration on Day 1 17 2 Vehicle In vivo Gene 4 mMsolution in PBS Shuttle (DOTAP + Cholesterol) 18 6-8 Ad-βgal In vivoGene 0.65 × 10¹¹   4 mM of In vivo Gene Shuttle (DOTAP + Shuttle mixedwith Cholesterol) virus. Administration on Day 1. (Dilute 60 ml of Lipidwith 90 ml of water. Then add 150 ul of Ad-bgal) 19 2 Vehicle 0.5%Polidocanol 0.5% Polidocanol in PBS-10% glycerol on Day 1 20 6-8 Ad-βgal0.5% Polidocanol 1.3 × 10¹¹   0.5% Polidocanol pretreatment followed byvirus administration on Day 1 21 2 Vehicle 0.4% HPMC 2910 0.4% HPMC 2910in PBS-10% glycerol on Day 1 22 6-8 Ad-βgal 0.4% HPMC 2910 0.5 × 10¹¹  0.8% HPMC 2910 mixed with an equal volume of the virus and thenadministered on Day 1 23 2 Vehicle 100 mg/ml Poly- 100 ug/ml Poly-LysineLysine in PBS-10% glycerol on Day 1 24 6-8 Ad-βgal 100 mg/ml Poly- 0.5 ×10¹¹   200 ug/ml Poly-Lysine Lysine mixed with an equal volume of thevirus and then administered on Day 1 25 2 Vehicle 0.1% n-dodecyl-b- 0.1%n-dodecyl-b-D D glucopyranoside glucopyranoside in PBS-10% glycerol onDay 1 26 6-8 Ad-βgal 0.1% n-dodecyl-b- 1 × 10¹¹ 0.1% n-dodecyl-b-D Dglucopyranoside glucopyranoside pretreatment followed by virusadministration on Day 1 27 2 Vehicle 0.4% PVA 0.4% PVA in PBS- 10%glycerol on Day 1 28 6-8 Ad-βgal 0.4% PVA 0.5 × 10¹¹   0.8% PVA mixedwith an equal volume of the virus and then administered on Day 1 29 2Vehicle 0.4% PVP 0.4% PVP in PBS- 10% glycerol on Day 1 30 6-8 Ad-βgal0.4% PVP 0.5 × 10¹¹   0.8% PVP mixed with an equal volume of the virusand then administered on Day 1 31 2 Vehicle 0.1% Cholesterol- 0.1%Cholesterol- Cyclodextrin Cyclodextrin reagent reagent in PBS-10%glycerol on Day 1 32 6-8 Ad-βgal 0.1% Cholesterol- 0.5 × 10¹¹   0.2%Cholesterol- Cyclodextrin Cyclodextrin reagent reagent mixed with anequal volume of the virus and then administered on Day 1 33 2 Vehicle0.05% n-Dodecyl 0.05% n-Dodecyl b-D- b-D-Maltoside Maltoside in PBS-10%glycerol on Day 1 34 6-8 Ad-βgal 0.05% n-Dodecyl 1 × 10¹¹ 0.05%n-Dodecyl b-D- b-D-Maltoside Maltoside pretreatment followed by virusadministration on Day 1 35 2 Vehicle 0.3% Benzyl 100 μl of 0.3% BenzylAlcohol Alcohol in PBS-10% glycerol on Day 1 36 6-8 Ad-βgal 0.3% Benzyl1 × 10¹¹ 0.3% Benzyl Alcohol Alcohol pretreatment followed by virusadministration on Day 1 37 2 Vehicle 0.1% Benzyl 100 μl of 0.1% BenzylAlcohol Alcohol in PBS-10% glycerol on Day 1 38 6-8 Ad-βgal 0.1% Benzyl1 × 10¹¹ 0.1% Benzyl Alcohol Alcohol pretreatment followed by virusadministration on Day 1 39 2 Vehicle 0.1% 0.1% OxychloroseneOxychlorosene in PBS-10% glycerol on Day 1 40 6-8 Ad-βgal 0.1% 1 × 10¹¹0.1% Oxychlorosene Oxychlorosene pretreatment (5 min) followed by virusadministration on Day1 41 2 Vehicle 0.4% 0.4% OxychloroseneOxychlorosene in PBS-10% glycerol on Day 1 42 6-8 Ad-βgal 0.4% 1 × 10¹¹0.4% Oxychlorosene Oxychlorosene pretreatment (5 min) followed by virusadministration on Day1 43 2 Vehicle 0.02% Polidocanol 0.02% Polidocanolin PBS-10% glycerol on Day 1 44 6-8 Ad-βgal 0.02% Polidocanol 1 × 10¹¹0.02% Polidocanol pretreatment followed by virus administration on Day 145 2 Vehicle 0.2% Polidocanol 0.2% Polidocanol in PBS-10% glycerol onDay 1 46 6-8 Ad-βgal 0.2% Polidocanol 1 × 10¹¹ 0.2% Polidocanolpretreatment followed by virus administration on Day 1 47 2 Vehicle0.02% n-Dodecyl 0.02% n-Dodecyl b-D- b-D-Maltoside Maltoside in PBS-10%glycerol on Day 1 48 6-8 Ad-βgal 0.02% n-Dodecyl 1 × 10¹¹ 0.02%n-Dodecyl b-D- b-D-Maltoside Maltoside pretreatment followed by virusadministration on Day1 49 2 Vehicle 0.2% n-Dodecyl b- 0.2% n-Dodecylb-D- D-Maltoside Maltoside in PBS-10% glycerol on Day 1 50 6-8 Ad-βgal0.2% n-Dodecyl b- 1 × 10¹¹ 0.2% n-Dodecyl b-D- D-Maltoside Maltosidepretreatment followed by virus administration on Day1 51 2 Vehicle 0.2%sodium salt 0.2% sodium salt of of Dodecyl Dodecyl benzenesulfonicbenzenesulfonic acid acid in PBS-10% glycerol on Day 1 52 6-8 Ad-βgal0.2% sodium salt 1 × 10¹¹ 0.2% sodium salt of of Dodecyl Dodecylbenzenesulfonic benzenesulfonic acid acid pretreatment followed by virusadministration on Day 1 53 2 Vehicle 0.1% sodium 0.1% sodium dodecyldodecyl sulphate sulphate in PBS-10% glycerol on Day 1 54 6-8 Ad-βgal0.1% sodium 1 × 10¹¹ 0.1% sodium dodecyl dodecyl sulphate sulphatepretreatment followed by virus administration on Day1 55 2 Vehicle 0.1%Tween 20 0.1% Tween 20 in PBS-10% glycerol on Day 1 56 6-8 Ad-βgal 0.1%Tween 20 1 × 10¹¹ 0.1% Tween 20 pretreatment followed by virusadministration on Day 1 57 2 Vehicle 0.1% Triton X-100 0.1% Triton X-100in PBS-10% glycerol on Day 1 58 6-8 Ad-βgal 0.1% Triton X-100 1 × 10¹¹0.1% Triton X-100 pretreatment followed by virus administration on Day 159 2 Vehicle 0.1% Forlan C-24 0.1% Forlan C-24 in (PEG Cholesterol)PBS-10% glycerol on Day 1 60 6-8 Ad-βgal 0.1% Forlan C-24 1 × 10¹¹ 0.1%Forlan C-24 (PEG Cholesterol) pretreatment followed by virusadministration on Day 1 61 2 Vehicle 0.1% Decyl-b-D- 0.1% Decyl-b-D-Maltoside Maltoside in PBS-10% glycerol on Day 1 62 6-8 Ad-βgal 0.1%Decyl-b-D- 1 × 10¹¹ 0.1% Decyl-b-D- Maltoside Maltoside pretreatmentfollowed by virus administration on Day1 63 2 Vehicle 0.1% 6- 0.1% 6-Cyclohexylhexyl- Cyclohexylhexyl-b-D- b-D-Maltoside Maltoside in PBS-10%glycerol on Day 1 64 6-8 Ad-βgal 0.1% 6- 1 × 10¹¹ 0.1% 6-Cyclohexylhexyl- Cyclohexylhexyl-b-D- b-D-Maltoside Maltosidepretreatment followed by virus administration on Day1 65 2 Vehicle 0.1%Tromboject 0.1% Tromboject in (Sodium PBS-10% glycerol on TetradecylSulfate) Day 1 66 6-8 Ad-βgal 0.1% Tromboject 1 × 10¹¹ 0.1% Tromboject(Sodium pretreatment followed Tetradecyl Sulfate) by virusadministration on Day 1 67 2 Vehicle 0.1% Phenyl B-D- 0.1% Phenyl B-D-Glucopyranoside Glucopyranoside in PBS-10% glycerol on Day 1 68 6-8Ad-βgal 0.1% Phenyl B-D- 1 × 10¹¹ 0.1% Phenyl B-D- GlucopyranosideGlucopyranoside pretreatment followed by virus administration on Day 169 2 Vehicle 0.1% Sucrose 0.1% Sucrose Monolaurate Monolaurate in PBS-10% glycerol on Day 1 70 6-8 Ad-βgal 0.1% Sucrose 1 × 10¹¹ 0.1% SucroseMonolaurate Monolaurate pretreatment followed by virus administration onDay 1 71 2 Vehicle 0.1% 1-O-dodecyl- 0.1% 1-O-dodecyl- rac-glycerolrac-glycerol in PBS- 10% glycerol on Day 1 72 6-8 Ad-βgal 0.1%1-O-dodecyl- 1 × 10¹¹ 0.1% 1-O-dodecyl- rac-glycerol rac-glycerolpretreatment followed by virus administration on Day 1The concentration of Ad-βgal virus for the data in Table 2 was 1.3×10¹²vp/ml (1st preparation, particle: PFU: 30) and 1×10¹² vp/ml (2^(nd)preparation, particle: PFU: 30) as determined by optical densitymeasurements.

Treatment Procedure

1. The animals were anesthetized with isoflurane and a 24 g catheter wasintroduced through the urethra into the bladder.

2. Residual urine was emptied and the bladder was flushed 3 times with100 μl each of PBS.

3. Based on the reagent being tested, bladder pretreatment was performedas follows:

Poloxomer 407 procedure: Washed 2 times with 100 μl each. Retained the3^(rd) wash for 5 min and gave one additional wash. Performed 3 timesPBS wash prior to virus instillation.

Poloxomer 188 procedure: Washed 2 times with 100 μl each. Retained the3^(rd) wash for 5 minutes and gave one additional wash. Performed 3times PBS wash prior to virus instillation.

Lipofectamine 2000 procedure: Added 5 μl of stock Lipofectamine (1mg/ml) to 195 μl of PBS-10% glycerol. Mixed with an equal volume ofAd-βgal virus and incubated for 15 minutes. Administered 100 μl of themixture intravesically and retained in the bladder for 30 minutes.

Benzyl Alcohol procedure: Washed 2 times with 100 μl each. Retained the3^(rd) wash for 15 minutes and then gave one additional wash. Performed3 times PBS wash prior to virus instillation.

Oxychlorosene procedure: Washing performed as mentioned in the doseregimen (i.e., 3 washes of 100 μl each, one wash but retained for 5min., one wash but retained for 15 min). Performed 3 times PBS washprior to virus instillation.

Polidocanol procedure: Washed 2 times with 100 μl each. Retained the 3rdwash for 5 min and then gave one additional wash. Performed 3 times PBSwash prior to virus instillation.

DC-Cho procedure: Washed 2 times with 100 μl each. Retained the 3^(rd)wash for 5 min and then gave one additional wash. Performed 3 times PBSwash prior to virus instillation.

0.4% HPMC 2910 procedure: No pretreatment. An equal volume of the viruswas mixed with 0.8% solution of HPMC2910 and the mixture was instilledinto the bladder for 30 minutes.

100 mg/ml Poly-Lysine procedure: No pretreatment. An equal volume of thevirus was mixed with 100 mg/ml solution of Poly-Lysine and the mixturewas instilled into the bladder for 30 minutes.

0.4% polyvinyl alcohol (PVA) procedure: No pretreatment. An equal volumeof the virus was mixed with 0.8% solution of PVA and the mixture wasinstilled into the bladder for 30 minutes.

n-dodecyl-β-D glucopyranoside procedure: Washed 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

0.4% PVP procedure: No pretreatment. An equal volume of the virus wasmixed with 0.8% solution of PVP and the mixture was instilled into thebladder for 30 min.

0.1% cholesterol-cyclodextrin reagent procedure: No pretreatment. Anequal volume of the virus was mixed with 0.2% solution ofCholesterol-Cyclodextrin and the mixture was instilled into the bladderfor 30 minutes.

n-dodecyl-β-D-maltoside procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

Sodium salt of dodecyl benzenesulfonic acid procedure: Washed 2 timeswith 100 μl each. Retained the 3^(rd) wash for 5 min and then gave oneadditional wash. Performed 3 times PBS wash prior to virus instillation.

0.1% sodium dodecyl sulphate procedure: Wash 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

0.1% Tween 20 procedure: Washed 2 times with 100 μl each. Retained the3^(rd) wash for 5 min and then gave one additional wash. Performed 3times PBS wash prior to virus instillation.

0.1% Triton® X-100 procedure: Washed 2 times with 100 μl each. Retainedthe 3^(rd) wash for 5 min and then gave one additional wash. Perform 3times PBS wash prior to virus instillation.

0.1% Forlan C-24 procedure: Washed 2 times with 100 μl each. Retainedthe 3^(rd) wash for 5 min and then gave one additional wash. Performed 3times PBS wash prior to virus instillation.

0.1% decyl-b-D-maltoside procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

0.1% 6-cyclohexylhexyl-b-D-maltoside procedure: Washed 2 times with 100μl each. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

0.1% sodium tetradecyl sulfate (Tromboject®, Omega Laboratories Ltd.)procedure: Washed 2 times with 100 μl each. Retained the 3^(rd) wash for5 min and then gave one additional wash. Performed 3 times PBS washprior to virus instillation.

0.1% phenyl-β-D-glucopyranoside procedure: Washed 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

0.1% sucrose monolaurate procedure: Washed 2 times with 100 μl each.Retained the 3^(rd) wash for 5 min and then gave one additional wash.Performed 3 times PBS wash prior to virus instillation.

0.1% 1-O-dodecyl-rac-glycerol procedure: Washed 2 times with 100 μleach. Retained the 3^(rd) wash for 5 min and then gave one additionalwash. Performed 3 times PBS wash prior to virus instillation.

In vivo geneSHUTTLE™ procedure. Mixed 4 mM of In vivo geneSHUTTLE™ withvirus. Administration on Day 1. Diluted 60 ml of Lipid with 90 ml ofwater. Then added 150 μl of Ad-βgal.

4. Virus treatment (45 min) stopped by withdrawing the virus andflushing the bladders 3 times with 100 μl of PBS.

Measurement/Determinations

The clinical condition of the animals were observed before dosing on theday of treatment, and animals were observed daily during theexperimental period.

Assessment of β-Galactosidase Activity

Animals were killed 48 hours after treatment. The bladders were filledwith 0.1-ml whole organ fixative: 2% Neutral buffered formalin, 2%glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4. The bladders were thenremoved and immersed in whole organ fixative for 1 hour. Thereafter,each bladder was cut open longitudinally, rinsed (in 2 mM MgCl₂, 0.1%deoxycholate, 0.2% Triton) for 24 hours at 4° C., and submerged intoX-gal staining solution. Transgene expression in the luminal epitheliumof the longitudinally opened bladders was empirically determined.

Histopathology

Bladders fixed in whole organ fixative were sectioned and stained withhematoxylin-eosin for histologic examination.

Results

The results of the above experiments can be summarized as follows:

Pre-treatment of the bladder with 4% Poloxamer 407 (Pluronic 127) for 5minutes resulted in <5% transduction.

Treatment of the bladder with a lipofectamine and virus mixture (nopretreatment) resulted in <5% transduction.

Treatment of the bladder with an In vivo geneSHUTTLE™ and virus mix (nobladder pretreatment) resulted in <5% transduction.

A pre-treatment of the bladder with 0.1% oxychlorosene for 5 minutesresulted in >90% transduction of the urothelium. The pathologists reportindicated mild submucosal edema with intact epithelial layer.

A pre-treatment of the bladder with 0.2% oxychlorosene for 5 minutesresulted in >90% transduction of the urothelium. The pathologists reportindicated minimal submucosal edema and perivascular lymphocytes.

A pre-treatment of the bladder with 0.2% oxychlorosene for 15 minutesresulted in >90% transduction of Urothelium. The pathologists reportindicated focal severe ulceration with suppurtative exudate, hemorrhageand edema in the submucosa.

A pre-treatment of the bladder with 0.4% oxychlorosene for 5 minutesresulted in >90% transduction of Urothelium. The pathologists reportindicated moderate submucosal edema with focal large ulcer.

A pre-treatment of the bladder with 0.02% polidocanol for 5 minutesresulted in 10-20% transduction of the urothelium. The pathologistsreport indicated an intact mucosa.

A pre-treatment of the bladder with 0.05% polidocanol for 5 minutesresulted in 30-40% transduction of the urothelium. The pathologistsreport indicated minimal submucosal edema.

A pre-treatment of the bladder with 0.2% polidocanol for 5 minutesresulted in 50-80% transduction of Urothelium. The pathologists reportindicated erosions and focal ulcer as well as mucosal compromise.

A pre-treatment of the bladder with 0.02% n-dodecyl β-D-maltoside for 5minutes resulted in 50-80% transduction of the urothelium. Thepathologists report indicated no significant lesions.

A pre-treatment of the bladder with 0.05% n-dodecyl β-D-maltoside for 5minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated no significant lesions.

A pre-treatment of the bladder with 0.2% n-dodecyl β-D-maltoside for 5minutes resulted in >90% transduction of the urothelium. Thepathologists report indicated erosions, focal ulcer, moderate submucosaledema with mucosal compromise.

A pre-treatment of the bladder with 0.2% dodecyl benzenesulfonic acidfor 5 minutes resulted in 20-40% transduction of the urothelium.

As can be seen from the above results, several single compounds and onemixed reagent showed significantly increased transduction as measured bythe levels of final blue stain (LacZ). Several other single compoundsresulted in enhanced but smaller levels of transduction. An ethanolpre-treatment was used as a reference to validate each chemical tested.Even with an ethanol percentage as high as 30%, only 10-20% transductionwas observed. The “strong responders” were those transduction enhancingagents which exhibited significantly better (i.e., 70-90% staining) thanthe ethanol pre-treatment controls, which exhibited 10-20% staining. Theweak responders had significantly less stained area compared to theethanol control group.

The strongest response (i.e., highest level of transduction) wasobserved following pretreatment of the bladder surface with: 0.02%-0.5%polidocanol; 0.02-0.5% n-dodecyl-b-D-maltoside; 0.1%6-cyclohexylhexyl-b-D-maltoside; 0.1%-0.4% oxychlorosene; 0.2% sodiumsalt of dodecyl benzenesulfonic acid; and 0.1% sodium dodecyl sulphate.

The “weak responders” included 0.1% decyl-b-D-maltoside and 0.1% Triton®X-100.

Although not wishing to be bound by theory, the mechanism of action canbe hypothesized by analyzing the physical and chemical properties ofsuccessful transduction enhancing reagents. The transduction enhancingreagent in general is a surfactant. The surfactant can be ionic ornon-ionic. The surfactant preferably has both hydrophilic and lipophilicsections. The hydrophilic portion of the molecule contributes to watersolubility while the lipophilic (i.e., hydrophobic) portion helpsmolecular interactions with lipids. The hydrophilic/lipophilic balanceor HLB ratio is an indication of the relative size of each part of themolecule.

Sugar Based Surfactants (Saccharides)

The transduction enhancing agent according to the invention can be asugar (e.g., a mono-, di-, or poly-saccharide) having a lipophilicsubstituent. The transduction enhancing agent can be any mono-, di-, orpoly-saccharide having a lipophilic substituent. According to apreferred embodiment of the invention, the transduction enhancing agentis a di-saccharide having a lipophilic substituent. Exemplarydi-saccharides include maltose or sucrose. Other di-saccharides havinglipophilic substituents, however, can also be used including lactose,isomaltose, trehalose or cellobiose.

The lipophilic substituent can be linear (e.g., a straight chainn-alkane or alkene) or non-linear (e.g., cyclic or branched chainalkanes or alkenes). The lipophilic substituent can also be an alkanoicacid residue. The length of the lipophilic substituent can be varied toachieve the desired hydrophilic-lipophilic balance. Tests on variousmaltoside substituted compounds indicated that a sufficient lipophiliclength resulted in improved transduction efficacy. For example, bothn-dodecyl-β-D-maltoside and 6-cyclohexylhexyl-β-D-maltoside increasedtransduction significantly. In contrast, n-decyl-β-D-maltoside had onlya slight effect on transduction.

Results for bladder pretreatment with n-dodecyl-β-D-maltoside are shownin FIGS. 25-29. FIG. 25A is a photograph showing the luminal surface ofa first murine bladder after pretreatment with a 0.02% n-dodecylβ-D-maltoside solution followed by infection with Ad-LacZ. FIGS. 25B and25C are photographs showing the cross section of the murine bladder ofFIG. 25A. FIG. 25B was taken at 40X and FIG. 25C was taken at 100×magnification. FIG. 26A is a photograph showing the luminal surface of asecond murine bladder after pretreatment with a 0.02% n-dodecylβ-D-maltoside solution followed by infection with Ad-LacZ. FIGS. 26B and26C are photographs showing the cross section of the murine bladder ofFIG. 26A. FIG. 26B was taken at 40× and FIG. 26C was taken at 100×magnification. FIG. 27A is a photograph showing the luminal surface of afirst murine bladder after pretreatment with a 0.05% n-dodecylβ-D-maltoside solution followed by infection with Ad-LacZ. FIGS. 27B and27C are photographs showing the cross section of the murine bladder ofFIG. 27A. FIG. 27B was taken at 40× and FIG. 27C was taken at 100×magnification. FIG. 28A is a photograph showing the luminal surface of afirst murine bladder after pretreatment with a 0.05% n-dodecylβ-D-maltoside solution followed by infection with Ad-LacZ. FIGS. 28B and28C are photographs showing the cross section of the murine bladder ofFIG. 28A. FIG. 28B was taken at 40× and FIG. 28C was taken at 100×magnification. FIG. 29A is a photograph showing the luminal surface of afirst murine bladder after pretreatment with a 0.2% n-dodecylβ-D-maltoside solution followed by infection with Ad-LacZ. FIGS. 29B and29C are photographs showing the cross section of the murine bladder ofFIG. 29A. FIG. 29B was taken at 40× and FIG. 29C was taken at 100×magnification.

The chemical formula for n-dodecyl-β-D-maltoside andn-decyl-β-D-maltoside is given below:

where n is 11 and 9, respectively. The chemical formula for6-cyclohexylhexyl-β-D-maltoside is:

where n is 6.

The transduction experiments demonstrated that a small reduction in thesize of the lipophilic side chain (i.e., CH₂—CH₂) can limit the efficacyof the molecule for transduction enhancement to a great degree. It isimportant to note that all of the above compounds had good solubility inboth water and PBS buffer.

Compounds in this class of surfactants having a shorter hydrophilicmoiety were also evaluated. The results forn-dodecyl-β-D-glucopyranoside showed little or no enhancement oftransduction. The chemical formula for n-dodecyl-β-D-glucopyranoside is:

where n is 11. While not wishing to be bound by theory, the relativesizes of the hydrophilic and lipophilic portions of the molecule appearto influence transduction enhancement. Therefore, shorter chainn-alkyl-β-D-glucopyranosides (e.g., n-hexyl-β-D-glucopyranoside) mayexhibit improved transduction.

Any mono-, di-, or poly-saccharide having a lipophilic substituent canbe used as a transduction enhancing agent according to the invention.Exemplary di-saccharide compounds include sucrose, lactose, maltose,isomaltose, trehalose, and cellobiose. The lipophilic substituentpreferably comprises an alkyl or alkenyl group. According to a preferredembodiment of the invention, the lipophilic substituent is an alkanoicacid residue.

Although the β-forms of the mono- and di-saccharides are describedabove, the α-forms of these and other mono-, di-, or poly-saccharidecompounds can also be used according to the invention. Exemplaryα-saccharide transduction enhancing agents according to the inventioninclude n-dodecyl-α-D-maltoside, n-hexyl-α-D-glucopyranoside and6-cyclohexylhexyl-α-D-maltoside. Additionally, either the D- or L-formsof the mono-, di-, or poly-saccharides may be used as transductionenhancing agents according to the invention.

FIGS. 31A-31E are photographs showing the luminal surfaces of murinebladders treated with alkyl maltoside and alkyl maltopyranosidepretreating agents having various length alkyl side chains. FIG. 31A isa photograph of the luminal surface of a murine bladder treated withn-dodecyl-β-D-maltoside (C12 alkyl side chain) prior to infection withAd-LacZ (10⁹ vp). FIG. 31B shows the luminal surface of a murine bladdertreated with tridecyl-β-D-maltopyranoside (C13 alkyl side chain) priorto infection. FIG. 31C shows the luminal surface of a murine bladdertreated with n-tetradecyl-β-D-maltoside (C14 alkyl side chain) prior toinfection. FIG. 31D shows the luminal surface of a murine bladdertreated with n-decyl-β-D-maltoside (C10 alkyl side chain) prior toinfection. FIG. 3 1E shows the luminal surface of a murine bladdertreated with n-octyl-β-D-maltopyranoside (C8 alkyl side chain) prior toinfection.

Ionic Alkyl Surfactants

Ionic alkyl surfactants can also be used as a transduction enhancingcompounds according to the invention. Exemplary ionic alkyl surfactantsinclude sodium dodecyl sulfate which has a formula represented by:

Another exemplary ionic surfactant is the sodium salt ofdodecyl-benzenesulfonic acid which has a chemical formula representedby:

Surfactants of the above type were evaluated and were found to exhibitenhanced transduction comparable to the non-ionic reagents set forthabove. These results are shown in FIG. 30 for dodecyl benzenesulfonicacid sodium salt. As can be seen by FIGS. 30A-30C, dodecylbenzenesulfonic acid sodium salt, enhanced the transduction of Ad-LacZin murine bladders. FIG. 30A is a photograph showing the luminal surfaceof a first murine bladder after pretreatment with a 0.2% sodium salt ofdedecyl benzenesulfonic acid solution followed by infection withAd-LacZ. FIGS. 30B and 30C are photographs showing the cross section ofthe murine bladder of FIG. 30A. FIG. 30B was taken at 40× and FIG. 30Cwas taken at 100× magnification.

FIGS. 32A-32C are photographs showing the luminal surfaces of murinebladders treated with sodium alkyl sulfate pretreating agents havingvarious length alkyl chains. FIG. 32A shows the luminal surface of abladder treated with sodium dodecyl sulfate (SDS) (C12 alkyl side chain)prior to infection with Ad-LacZ (10⁹ vp). FIG. 32B shows the luminalsurface of a bladder treated with sodium decyl sulfate (C10 alkyl sidechain) prior to infection. FIG. 32C shows the luminal surface of abladder treated with sodium octyl sulfate (C8 alkyl side chain) prior toinfection.

The ionic alkyl surfactants consist of two portions, a hydrophilicportion and a lipophilic portion. The arrangement of these portions ofthe molecule is similar to the sugar-based enhancing agents describedabove. According to the invention, compounds similar to those set forthabove and having variations in alkyl substitution can also be used.

Alkyl(Ether) Alcohols

Also according to the invention, an alkyl ether compound can be used asa transduction enhancing compound. Polidocanol, an alkyl ether havingthe following chemical formula:

˜C₁₂H₆₂O—(CH₂—CH₂—O)₋₉

and a total formula of ˜C₃₀H₆₂O₁₀, was evaluated. The polidocanol usedin the evaluation was sold under the name Thesit®, which is a registeredtrademark of Desitin-Werk, Carl Klinke GmbH, Hamburg, Germany). Thereare several other chemical names for polidocanol such aspolyethyleneglycoldodecyl ether [9002-92-0], lauryl alcohol, andmacrogol lauryl ether.

Results for pretreatment of the bladder surface with variousconcentrations of polidocanol are shown in FIGS. 19-24. Results forpretreatment of the bladder surface with 0.02% polidocanol are shown inFIGS. 19 and 20. FIG. 19A is a photograph showing the luminal surface ofa first murine bladder after pretreatment with a 0.02% polidocanolsolution followed by infection with Ad-LacZ. FIGS. 19B and 19C arephotographs showing the cross section of the murine bladder of FIG. 19A.FIG. 19B was taken at 40× and FIG. 19C was taken at 100× magnification.FIG. 20A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.02% polidocanol solution followed byinfection with Ad-LacZ. FIGS. 20B and 20C are photographs showing thecross section of the murine bladder of FIG. 20A. FIG. 20B was taken at40× and FIG. 20C was taken at 100× magnification.

Results for pretreatment of the bladder surface with 0.05% polidocanolare shown in FIGS. 21 and 22. FIG. 21A and 21B are photographs showingthe outside and luminal surfaces, respectively, of a first murinebladder after pretreatment with a 0.05% polidocanol solution followed byinfection with Ad-LacZ. FIGS. 21C and 21D are photographs showing thecross section of the murine bladder of FIG. 21A. FIG. 21B was taken at40× and FIG. 21C was taken at 100× magnification. FIG. 22A and 22B arephotographs showing the outside and luminal surfaces, respectively, of asecond murine bladder after pretreatment with a 0.05% polidocanolsolution followed by infection with Ad-LacZ. FIGS. 22C and 22D arephotographs showing the cross section of the murine bladder of FIG. 22A.FIG. 22B was taken at 40× and FIG. 22C was taken at100× magnification.

Results for pretreatment of the bladder surface with 0.2% polidocanolare shown in FIGS. 23 and 24. FIG. 23A is a photograph showing theluminal surface of a first murine bladder after pretreatment with a 0.2%polidocanol solution followed by infection with Ad-LacZ. FIGS. 23B and23C are photographs showing the cross section of the murine bladder ofFIG. 23A. FIG. 23B was taken at 40× and FIG. 23C was taken at 100×magnification. FIG. 24A is a photograph showing the luminal surface of asecond murine bladder after pretreatment with a 0.2% polidocanolsolution followed by infection with Ad-LacZ. FIGS. 24B and 24C arephotographs showing the cross section of the murine bladder of FIG. 24A.FIG. 24B was taken at 40× and FIG. 24C was taken at 100× magnification.

Triton® X-100, having a general formula of:

wherein x=10 was also evaluated and was also found to enhancetransduction. A similar compound having a cyclohexane ring rather than abenzene ring can also be used as a transduction enhancing agentaccording to the invention. This compound has the following chemicalstructure:

wherein x=10. Compounds of the above type wherein x is any positiveinteger can also be used according to the invention.

Similar alkyl(ether) compounds having the general structure of:

are also commercially available. The trade name for these compounds is“Brij”. The compound shown above is designated “Brij 56”. Brij 56 hasthe chemical formula C₂₀H₄₂O₅. Another commercially available compound,“Brij 58”, has the chemical formula C₅₆H₁₁₄O₂₁.

Any of the above mentioned alkyl(ether) compounds can be used astransduction enhancing agents according to the invention.

Sodium Oxychlorosene

A composition comprising a sodium salt of dodecylbenzenesulfonic acidand hypochlorous acid (i.e., sodium oxychlorosene) at a pH of about 6.5to 6.9 was evaluated. The sodium oxychlorosene used in these evaluationswas sold under the name Clorpactin WCS-90 (manufactured by Guardian Labsand sold by Cardinal Health). Sodium oxychlorosene has been used totreat urinary tract infections and in abdominal and plastic surgery.

Results for pretreatment of the bladder surface with sodiumoxychlorosene are shown in FIGS. 8-18. FIGS. 8A-8N are photographsshowing seven murine bladders after pretreatment with a 0.2%oxychlorosene solution for 5 minutes followed by infection with Ad-LacZ.FIGS. 8A and 8B show the outside and luminal surfaces, respectively, ofthe first bladder, FIGS. 8C and 8D show the outside and luminalsurfaces, respectively, of the second bladder, FIGS. 8E and 8F show theoutside and luminal surfaces, respectively, of the third bladder, FIGS.8G and 8H show the outside and luminal surfaces, respectively, of thefourth bladder, FIGS. 8I and 8J show the outside and luminal surfaces,respectively, of the fifth bladder, FIGS. 8K and 8L show the outside andluminal surfaces, respectively, of the sixth bladder, and FIGS. 8M and8N show the outside and luminal surfaces, respectively, of the seventhbladder.

FIGS. 9A-9N are photographs showing seven murine bladders afterpretreatment with a 0.2% oxychlorosene solution for 15 minutes followedby infection with Ad-LacZ. FIGS. 9A and 9B show the outside and luminalsurfaces, respectively, of the first bladder, FIGS. 9C and 9D show theoutside and luminal surfaces, respectively, of the second bladder, FIGS.9E and 9F show the outside and luminal surfaces, respectively, of thethird bladder, FIGS. 9G and 9H show the outside and luminal surfaces,respectively, of the fourth bladder, FIGS. 9I and 9J show the outsideand luminal surfaces, respectively, of the fifth bladder, FIGS. 9K and9L show the outside and luminal surfaces, respectively, of the sixthbladder, and FIGS. 9M and 9N show the outside and luminal surfaces,respectively, of the seventh bladder.

FIGS. 10A and 10B are photographs showing the cross section of themurine bladders of FIGS. 8C and 8I, respectively. FIGS. 11A and 11B arephotographs showing the cross section of the murine bladders of FIGS. 9Cand 9I, respectively.

FIGS. 12A-12F are photographs showing the cross section of three murinebladders after pretreatment with a 0.2% oxychlorosene solution for 5minutes followed by infection with Ad-LacZ. FIGS. 12A and 12B arephotographs showing the cross-section of the first murine bladder, FIGS.12C and 12D are photographs showing the cross-section of the secondmurine bladder, and FIGS. 12E and 12F are photographs showing thecross-section of the third murine bladder. FIGS. 12A, 12C and 12E weretaken at 40× and FIGS. 12B, 12D and 12F were taken at 100×magnification.

FIGS. 13A-13F are photographs showing the cross section of three murinebladders after pretreatment with a 0.2% oxychlorosene solution for 15minutes followed by infection with Ad-LacZ. FIGS. 13A and 13B arephotographs showing the cross-section of the first murine bladder, FIGS.13C and 13D are photographs showing the cross-section of the secondmurine bladder, and FIGS. 13E and 13F are photographs showing thecross-section of the third murine bladder. FIGS. 13A, 13C and 13E weretaken at 40× and FIGS. 13B, 13D and 13F were taken at 100×magnification.

FIG. 14A is a photograph showing the luminal surface of a murine bladderafter pretreatment with a 0.1% oxychlorosene solution followed byinfection with Ad-LacZ. FIGS. 14B and 14C are photographs showing thecross section of the murine bladder of FIG. 14A. FIG. 14B was taken at40× and FIG. 14C was taken at 100× magnification;

FIG. 15A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.2% oxychlorosene solution followedby infection with Ad-LacZ. FIGS. 15B and 15C are photographs showing thecross section of the murine bladder of FIG. 15A. FIG. 15B was taken at40× and FIG. 15C was taken at 100× magnification.

FIG. 16A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.2% oxychlorosene solution followedby infection with Ad-LacZ. FIGS. 16B and 16C are photographs showing thecross section of the murine bladder of FIG. 16A. FIG. 16B was taken at40× and FIG. 16C was taken at 100× magnification.

FIG. 17A is a photograph showing the luminal surface of a first murinebladder after pretreatment with a 0.4% oxychlorosene solution followedby infection with Ad-LacZ. FIGS. 17B and 17C are photographs showing thecross section of the murine bladder of FIG. 17A. FIG. 17B was taken at40× and FIG. 17C was taken at 100× magnification.

FIG. 18A is a photograph showing the luminal surface of a second murinebladder after pretreatment with a 0.4% oxychlorosene solution followedby infection with Ad-LacZ. FIGS. 18B and 18C are photographs showing thecross section of the murine bladder of FIG. 18A. FIG. 18B was taken at40× and FIG. 18C was taken at 100× magnification.

Polymers with Alternating Hydrophilic and Lipophilic Units

Polymeric compounds comprising repeating sequences of alternating oridentical monomers were also tested. One such compound tested wasPoloxamer 407 (Pluronic 127) having a structure represented by thefollowing formula:

Poloxamers polymers come in a wide range of HLB values. Both of thecompounds tested, however, had only a minimal effect on the transductionof adenovirus. While not wishing to be bound by theory, it is believedthat compounds having separated, longer hydrophilic and lipophilicchains are more effective at enhancing transduction of the bladderepithelium.

Additional Transduction Enhancing Compounds

Additional compounds can also be used as transduction enhancing agentsaccording to the invention.

These compounds include ω-undecylenyl-β-D-maltopyranoside, which has astructure represented by:

Sugar based thiolic compounds such as alkyl-β-D-thioglucopyranosideshaving a general structure represented by:

may also be employed.

Additionally, alkyl-β-D-thiomaltopyranosides having a general structurerepresented by:

may also be used as transduction enhancing compounds according to theinvention.

Further, compounds having a positive charge such as

can also be used.

Additionally, compounds wherein the lipophilic and hydrophilic parts areconnected via a carboxylic bond can also be employed. An exemplarycompound of this type is6-O-methyl-n-heptylcarboxyl-α-D-glucopyranoside:

Sugar based compounds having alkyl groups with side groups or othermodifications may also be used. Exemplary compounds of this type include2-propyl-1-pentyl-β-D-maltopyranoside having a structure represented by:

Sarcosine compounds may also be used as transduction enhancing agentsaccording to the invention. Exemplary sarcosine compounds include sodiumalkyl sarcosine having a structure represented by:

Various substituted sugars can also be used as transduction enhancingcompounds. An exemplary substituted sugar which can be used as atransduction enhancing compound is a sucrose mono alkyl ester having achemical structure represented by:

Exemplary compounds of this type include compounds wherein n−10 (i.e.,sucrose monolaurate).

Also according to the present invention, methods of treating the luminalsurface of the bladder are provided. According to a preferred embodimentof the invention, the bladder is treated by instillation using bladdercatheterization. According to this embodiment, any urine in the bladderis first removed and the bladder is optionally washed with a buffer(e.g., PBS). A composition comprising the transduction enhancing agentis then applied to the luminal surface of the bladder (e.g., byinstillation). The transduction enhancing solution may be incubated forsome specified time or drained immediately. Multiple treatments with thecomposition comprising the transduction enhancing agent can beperformed. After treatment with the transduction enhancing agent, theluminal surface of the bladder may be washed with a buffer (e.g., PBS).A solution comprising the adenovirus can then be introduced into thebladder (e.g., by instillation). The solution comprising the adenoviruscan be removed immediately or, alternatively, the solution can beallowed to incubate for a certain amount of time. After treatment withthe adenovirus, the bladder surface can again be washed with a buffersolution (e.g., PBS). According to a preferred embodiment of theinvention, about 50 to about 500 ml of the transduction enhancingcomposition is delivered to the bladder by instillation for eachtreatment.

Alternatively, a composition comprising the transduction enhancing agentand the adenovirus can be used to treat the luminal bladder surface.According to this embodiment of the invention, any urine in the bladderis first removed and the bladder is then optionally washed with a buffer(e.g., PBS). A composition comprising the transduction enhancing agentand the adenovirus is then applied to the luminal surface of thebladder. The solution may be incubated for some specified time ordrained immediately. After treatment, the luminal surface of the bladdermay again be washed with a buffer (e.g., PBS).

Although phosphate buffered saline (PBS) is the preferred buffer, anyother pharmaceutical buffer can be used according to the invention.Exemplary buffers include sodium phosphate/sodium sulfate, Tris buffer,glycine buffer, sterile water and other buffers known in the art,including those described by Good, et al., Biochemistry 5, 467 (1966).The pH of the buffer can be in the range of 6.4 to 8.4, preferably 7 to7.5, and most preferably 7.2 to 7.4.

The composition comprising the transduction enhancing agent according tothe invention preferably also comprises an oxidizing agent. Exemplaryoxidizing agents include, but are not limited to, chlorite compounds,hypochlorous acid, hydrogen peroxide, and peroxyacetic acid. Accordingto a preferred embodiment of the invention, any of the single compoundtransduction enhancing agents can be combined with an oxidizing agentand used as a transduction enhancing agent.

As set forth above, the viral gene therapy vehicle can be an oncolyticvirus, for example an oncolytic adenovirus exemplified herein by CG8840.The adenovirus composition can further comprise a chemotherapeutic agentsuch as Docetaxel. The adenovirus composition preferably comprises fromabout 1×10¹¹ to about 1×10¹⁴ viral particles.

Various additional studies were conducted and are described below. Inthese studies, all percent values that are presented are weight percentvalues unless otherwise indicated.

Effects of Adenovirus Formulation with Different Concentrations ofDodecyl-β-D-Maltoside on the Infectivity of Mice Bladder Urothelium

-   Species: Female Balb/c Mouse (Taconic Laboratory)-2/group-   Study Design: To test the effect of formulating Ad-βgal virus with    different concentrations of Dodecyl-β-D-Maltoside and the resultant    infection of mice bladder urothelium

Dose/Route (Viral Particles #/Dose):

1×10¹⁰ vp/dose of Ad.CMV.LacZ (Lot#1351.122)

0.4%, 0.2%, 0.1%, and 0.05% of n-Dodecyl-β-D-Maltoside (Lot#100K5308).

An equal volume of 2× DDM and 2× Ad.CMV.LacZ was mixed togetherimmediately before instillation into the bladder. 100 μ of the mixturewas instilled into the bladder for 10 min, 20 min, and 45 min.

-   Endpoints: Bladders were harvested 48 hrs post virus infection with    0.1 ml whole organ fixative (2% Neutral buffered formalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: Formulating Ad-βgal virus with different concentrations of    Dodecyl-β-D-Maltoside resulted in a linear Adenovirus transduction    rate (0.1% >0.05% >0.025%). For the 20 min. instillation, 0.1% DDM    formulated with Ad-βgal virus resulted in about 80% gene expression    in bladder, while 0.05% DDM resulted in about 40% gene expression.    But with 45 min. instillation, all animals with 0.05%-0.2% DDM with    Ad-βgal virus had 100% gene expression in mice bladder. A 10 min.    instillation for this formulation method, however, did not achieve    an acceptable transduction rate. It was also found that gene    transduction could be achieved with a 10 min. virus instillation    after DDM pretreatment.

This study illustrates that DDM can be formulated with Ad-βgal virus inmice bladder model. Further, with a DDM pretreatment, the time for virusinstillation can be decreased to 10 min. from 45 min.

Effects of Different Diluents on the Infectivity of Mice BladderUrothelium by Adenovirus

-   Species: Female Balb/c Mouse (Taconic Laboratory)-2/group-   Study Design: To test the effects of different diluents on    Adenovirus infectivity of mice urothelium followed DDM pretreatment    (QQ5 minQ). The virus would be diluted 100 fold with the diluents    prior to instillation into mice bladder for 45 min.

Dose/Route:

1×10⁹ vp/dose of Ad.CMV.LacZ (Lot #1351.122)

Dodecyl-β-D-Maltoside (Lot #100K5308).

Diluents (A): 0.9% Sodium Chloride Injection Solution (BaxterLot#1A1322); (B): 2.5% Dextrose and 0.45% Sodium Chloride InjectionSolution (Baxter Lot# C529040); and (C): Plasma-lyte A InjectionSolution pH 7.4 (Baxter Lot# C558106); and ARCA buffer.

-   Endpoints: Bladders were harvested 48 hrs post virus infection with    0.1 ml whole organ fixative (2% Neutral buffered formalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: There were no significant differences in Ad-βgal gene    expression levels between mice bladder receiving virus diluted with    different diluents. All the animals had >90% in Ad-βgal gene    expression in mice bladder.    Adenovirus Dose Titration with Different Chemical Agents    Pretreatment on SD Rat Bladder Epithelium-   Species: Female Sprague Dawley Rat (Taconic Laboratory)-2/group-   Study Design: To test the virus infectivity of rat bladder    epithelium with different doses of Adenovirus following the    pretreatment with SDS or DDM. This study also explored relationship    between bladder volume and residual of SDS or DDM for adenovirus    infectivity.

Dose/Route:

4×10⁹ and 4×10¹⁰ vp/dose of Ad.CMV.LacZ (Lot#1351.122)

0.1% Dodecyl-β-D-Maltoside (Lot#100K5308)

-   0.1% SDS (Integra Lot#836011 and Lot# BK14J11)

400 μl of SDS or DDM was instilled into bladder (QQ5 minQ) followed bysix PBS washes, then Adenovirus for 15 min.

-   Endpoints: Bladders were harvested 48 hrs post virus infection with    0.2 ml whole organ fixative (2% Neutral buffered forrnalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: There were no significant differences in Ad-βgal gene    expression levels between rat bladders pretreated with different lot    of SDS and DDM. Rat bladders infected with 4×10¹⁰ vp adenovirus    achieved >90% Ad-βgal gene expression levels whereas rat bladders    infected with 4×10⁹ vp adenovirus had Ad-βgal gene expression levels    range from 30% to 50%. It was found that rat bladder needed 10 times    more adenovirus to achieve similar Ad-βgal gene expression levels    compared with mouse bladder. SDS and DDM pretreatment were both    effective to remove GAG layer of bladder epithelium.

Efficacy of Dodecyl-β-D-Maltoside as a Pretreatment Agent Prior toAdenovirus Infection of Mice Bladder Urothelium

-   Species: Female Balb/c Mouse (Taconic Laboratory)-2/group-   Study Design: To test DDM from two different suppliers as    pretreatment agents to enhance the adenovirus infection of mice    bladder urothelium in a large group of animals.

Dose/Route:

1×10¹⁰ vp/dose of Ad.CMV.LacZ (Lot #1351.122)

Dodecyl-p-D-Maltoside (Lot #018H7250 and Lot # P21/39/092)

100 μl of DDM was instilled into bladder (QQ5 minQ) followed by threePBS washes, then Adenovirus for 15 min.

-   Endpoints: Bladders were harvested 48 hrs post virus infection with    50 μl whole organ fixative (2% Neutral buffered formalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: There were no significant differences in Ad-βgal gene    expression levels between mice bladder pretreated with these two    different lots of DDM. In each case, >90% Ad-βgal expression was    achieved. Therefore, DDM from either manufacturer is a good    candidate for a chemical enhancer for adenovirus infection in mice    bladder.

Efficacy of Dodecyl-β-D-Maltoside as Chemical Enhancer Prior toAdenovirus Infection of SW780+Luc Orthotopic Bladder Tumor Model in Mice

-   Species: Female NCR nu/nu Mouse (Taconic Laboratory)-2/group-   Study Design: To show DDM pretreatments enhance Ad.Lac Z infectivity    in mice bladder urothelium. To further confirm the enhancer effect    on orthotopic tumors, two mice bearing orthotopic SW780 bladder    tumors were pretreated with or without DDM (QQ5 minQ) followed by 15    min Ad.Lac Z instillation. The Ad.Lac Z gene expression levels were    checked in these tumor cells 4 days post Ad.Lac Z infection.

Dose/Route:

1×10¹⁰ vp/dose. Ad.CMV.LacZ Lot#1351.122.

Dodecyl-β-D-Maltoside (Lot#018H7250).

100 μl of DDM was instilled into bladder (QQ5 minQ) followed by threePBS washes, then Adenovirus for 15 min.

-   Endpoints: Bladders were harvested 96 hrs post virus infection with    50 μl whole organ fixative (2% Neutral buffered formalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: There was a significant difference in Ad-βgal gene    expression levels between mice bladders pretreated with DDM and    control animals. Without DDM pretreatment, there was very low    Ad-βgal gene expression levels in bladder epithelium cells and tumor    cells. With DDM pretreatment, nearly 100% of the epithelium cells    were transduced with Ad-βgal gene in bladder tumors except those    dead necrotic tumor cells. It was concluded that DDM pretreatment    enhances Adenovirus infection in orthotopic tumor cells.

Testing of DDM and SDS Subgroup Compounds Containing Different Lengthsof Alkyl Side Chain and Different Types of Sugar Molecule asPretreatment Agents Prior to Adenovirus Infection of Mouse BladderUrothelium

-   Species: Female Balb/c Mouse (Taconic Laboratory)-3-2/group-   Study Design: Several compounds belonging to the    Dodecyl-β-D-Maltoside (DDM) and Sodium Dodecyl Sulfate (SDS)    subgroups containing different lengths of alkyl side chain and    different sugar molecules were tested as pretreatment agents to    enhance the adenovirus infection of mouse bladder epithelium. All    compounds were dissolved in PBS at 0.1% concentration.

Dose/Route:

1×10⁹vp/dose. Ad.CMV.LacZ (Lot #1351.122) 0.1% Dodecyl-β-D-Maltoside(Lot # P21/39/092) and 0.1% SDS (Lot #101K0036) were the positivecontrols for this study.

100 μl of Ad-βgal virus was instilled into bladder via intravesicleadministration for 15 min followed different chemicals pretreatment (QQ5minQ).

-   Endpoints: Bladders were harvested 48 hrs post virus infection with    50 μl whole organ fixative (2% Neutral buffered fornalin, 2%    glutaraldehyde, 2 mM MgCl₂, 10 mM PBS, pH 7.4).-   Results: For the DDM subgroup compounds, compounds with alkyl side    chain lengths longer than C12 had the best enhancer ability. DDM    subgroup compounds having C8, C10, C12, C13 and C14 alkyl side    chains were evaluated. For the SDS subgroup compounds, compounds    with alkyl side chain lengths shorter than C12 had very low Ad-βgal    gene expression levels. SDS subgroup compounds having C8, C10 and    C12 alkyl side chains were evaluated.

The type of sugar molecule did not appear to have a significant effecton the efficacy of these compounds as pretreatment enhancers with theexception of sucrose monolaurate. While not wishing to be bound bytheory, we believe that the disaccharide chain could enhance adenovirusinfectivity based on currently available data.

Pilot Efficacy Study: CG8840 Treatment with SW780+Luc Orthotopic BladderTumor Model in Mice

-   Species: Female NCR nu/nu Mouse (Taconic or Simonsen    Laboratory)-6/group for CG8840 and 3/group for CG7870-   Study Design: To test the efficacy of bladder specific oncolytic    virus CG8840 alone with prostate specific oncolytic virus CG7870.    Two treatment regimens were used for CG8840: 1×10¹⁰ and 1×10⁸    vp/dose. One treatment regimen was used for for CG7870: 1×10¹⁰    vp/dose. One dose of virus would be delivered into bladder weekly    for the consecutive three weeks.

Dose/Route:

1 million SW780+Luc cells Clone #19 (P4)

CG8840 Lot#1408.190; CG7870 (Lot #38.145)

0.1% Dodecyl-β-D-Maltoside (Lot #018H7250).

15 mg/ml Luciferin (Xenogen cat# XR-1001) substrate solution was made inPBS filtered with 0.2 μM filters.

120 μl of 1×10⁷ cell/ml SW780+luc cells were aliquot into each tube andkept in ice before instillation. One aliquot for each mice bladder.

-   Endpoints: Live image mice bladder was performed weekly for 10    weeks. H & E stain and Human Cytokeratin Stain were performed on    those available bladder and kidney samples.-   Results: There were significant differences in reducing tumor    volumes between CG8840 (1×10¹⁰ vp/dose) treated mice and control    animals (both CG7870 and no virus treated groups). Three out of six    mice treated with 1×10¹⁰ vp/dose CG8840 had completed tumor    regression after second dose instillation and kept tumor free until    the end of the study (week 10). Human cytokeratin stain showed there    were no tumor cells in those mouse bladders. Efficacy for 1×10⁸    vp/dose of CG8840 was not significant, there was a one out of five    mouse had incomplete tumor regression on week 5 and kept tumors at    bay to the end. Tumor metastasis to kidney is a significant threat    to tumor bearing mice survival. Any mice with kidney metastasis    would soon die even if they were free of bladder tumors. CG8840    Treatment with SW780+Luc Orthotopic Bladder Tumor Model in Mice-   Species: Female NCR nu/nu Mouse (Taconic Laboratory)-10/group-   Study Design: This study was designed to observe the efficacy of    bladder specific oncolytic virus CG8840 treatment modality for    bladder tumors. CG8840 virus was instilled into the bladder via    intravesical administration for 15 min and 30 min, respectively. To    reduced kidney tumor metastasis, two different DDM pretreatment    procedures were tested during SW780 tumor cell implanting: (1) 0.1%    DDM QQ5 minQ; and (2) 0.1% DDM Q10min.

Dose/Route:

1 million SW780+Luc cells Clone #19 (P4).

CG8840 Lot#1408.190. 0.1% Dodecyl-β-D-Maltoside (Lot #018H7250).

15 mg/ml Luciferin (Xenogen cat# XR-1001) substrate solution was made inPBS filtered with 0.2 μM filters.

90 μl of 1×10⁷cell/ml SW780+luc cells were aliquot into each tube andkept in ice before instillation. One aliquot for each mice bladder.

-   Endpoints: Live imaging of the mice bladders was performed weekly    for 8 weeks. H & E stain and Human Cytokeratin Stain were performed    on those available bladder and kidney samples.-   Results: There were significant difference in reducing tumor volumes    between CG8840 (15 min. and 30 min. virus treatment) treated mice    and control animals. In particular, seven out of nine mice with 15    min. virus treatment showed significant tumor volume regression    after first virus treatment. Five of them were tumor free till the    end of the study. The CG8840 treatment with three doses of 1×10¹⁰    vp/dose injected in three consecutive weeks proved very effective to    eradicate orthotopic SW780 tumors in mouse bladder. However, longer    virus instillation times (i.e., in excess of 15 min.) do not appear    to increase virus copy numbers within tumor cells.

For SW780 tumor cell implantation, 0.1% DDM pretreatment with Q10 min.washes, less kidney tumor metastasis was observed in mice withorthotopic tumors.

Varius additional studies were conducted to determine adenoviruscompatibility with various reagents. These studies are described below.

Study #1—Compatibility of Ad5-LacZ with Ethanol and Urine

In this study, each sample was incubated at 37° C. for one hour beforethe plaque assay started. The data for this study are shown below inTable 3.

TABLE 3 Adenovirus stability after incubation (EtOH, urine) TiterAverage Ratio Sample Name vp/mL pfu/mL Stdev. vp/pfu Ad5-Lac Z in ARCA1.30E+12 5.85E+10 1.49E+10 22.23 Ad5-Lac Z in ARCA 7.80E+11 6.36E+071.68E+07 12255.34 40% EtOH Ad5-Lac Z in ARCA 1.24E+12 5.07E+10 0.00E+0024.47 5% EtOH Ad5-Lac Z in ARCA 1.30E+10 6.26E+08 2.55E+08 20.75 1:100Urine Ad5-Lac Z −80° C. 1.30E+12 5.90E+10 9.28E+09 22.04 R-06-80 1 Hr.Hold 6.00E+11 2.34E+10 2.76E+09 25.65 R-06-81 6.00E+11 3.12E+10 8.27E+0919.24This data indicates that Ad-LacZ does not lose its activity afterincubation with urine, 5% ethanol, or ARCA at biological temperature.However, 40% ethanol inactivated most of the virus.Study #2: Compatibility of Ad5-LacZ with Ethanol

For this study, each sample was incubated at 37° C. before the plaqueassay started. The data for this study are shown below in Table 4.

TABLE 4 Adenovirus Stability after Incubation (EtOH, PBS) Titer AverageRatio Sample Name vp/mL pfu/mL St. dev. vp/pfu Ad 5 Lac Z 1.30E+124.34E+10 7.58E+09 30 Ad 5 Lac Z in PBS 1.30E+11 5.43E+09 4.26E+08 24PBS/10% EtOH 1.30E+11 3.72E+09 4.26E+08 35 PBS/20% EtOH 1.30E+111.81E+09 2.84E+08 72 PBS/30% EtOH 1.30E+11 <1.1E+5 pfu/mL 1230-0541.00E+11 9.55E+08 2.13E+08 105 R-06-80 6.00E+11 1.90E+10 6.20E+09 32The above results indicate that higher concentrations of ethanol (i.e.,≧10%) will result in partial to complete inactivation of adenovirus.Study #3—Compatibility of Adenovirus with Selected Enhancers

For this study, each sample was incubated at 37° C. or 25° C. before theplaque assay started. The data for this study are shown below in Table5.

TABLE 5 Adenovirus Stability after Incubation with Selected EnhancerSolutions Incub. Titer Average Ratio Solution Temp Time vp/mL pfu/mL St.Dev. vp/pfu None  5° C. none 4.23E+11 1.95E+10 0.00E+00 22 Oxychlorosene37° C. 1 hour 2.12E+11 8.82E+05 1.56E+05 240287 Dodecyl Maltoside 37° C.1 hour 2.12E+11 1.36E+10 1.38E+09 16 Polidocanol 37° C. 1 hour 2.12E+111.22E+10 2.07E+09 17 Oxychlorosene 25° C. 2 hours 2.12E+11 2.18E+080.00E+00 974 Dodecyl Maltoside 25° C. 2 hours 2.12E+11 1.32E+10 6.89E+0816 Polidocanol 25° C. 2 hours 2.12E+11 1.02E+10 4.82E+09 21 none −70°C.  none 6.00E+11 2.76E+09 2.13E+08 21.7

As can be seen from the above data, dodecyl maltoside and polidocanolare compatible with the adenovirus under the experimental conditionswhile oxychlorosene can inactivate the virus by increase in temperatureand time of incubation.

Study #4—Degradation of Adenovirus by Sodium Dodecyl Sulfate (SDS)

A series of experiments were preformed on samples of adenovirusincubated at 37° C. for 15-30 minutes in various concentrations of SDS.The resulting material,was examined by anion exchange chromatography. Itwas confirmed that concentrations of SDS above or equal to 0.025% SDSwill rapidly degrade the virus. SDS at 0.0125% did not reduce theparticle quality or quantity by AEX method after 30 minutes ofincubation.

Study #5—Degradation of Adenovirus by Sodium Dodecyl Sulfate (SDS)

In this study, adenovirus (Ad-LacZ) preparations (1.0×10¹² vp/ml) weremixed with solutions of SDS or DDM to obtain a mixture of adenovirus andeach of these surfactants. Final concentrations of SDS or DDM were 0.1%and the virus had a 5.0×10¹¹ vp/ml as the result of mixing. Thesesamples were incubated at 25° C. or 37° C. for one hour before freezing.They were sent to assay services for plaque assay analysis. The resultsfor this study are shown below in Table 6.

TABLE 6 Adenovirus Stability after Incubation with 0.1% SDS or 0.1% DDMSolutions Mean Titer Sample ID Replicate Titer PFU/mL Ad-LacZ 1351-122 13.70E+10 4.85E+10 2 6.00E+10 Ad-LacZ ARCA 1 Hr 25° C. 1 2.05E+102.05E+10 Ad-LacZ ARCA 1 Hr 37° C. 1 2.15E+10 2.15E+10 Ad-LacZ DDM 1 Hr25° C. 1 3.25E+10 3.25E+10 Ad-LacZ DDM 1 Hr 37° C. 1 3.55E+10 3.55E+10Ad-LacZ SDS 1 Hr 25° C. 1 no activity no activity Ad-LacZ SDS 1 Hr 37°C. 1 no activity no activityThis experiment confirmed that a relatively short exposure of adenovirusat elevated temperatures to SDS solutions will inactivate the virus.However, the DDM solution had no adverse effects on adenovirus.

The following table (Table 7) summarizes the transduction efficacy ofexemplary pretreatment agents. In Table 7, transduction efficacy isdefined as follows: “0” means 0-10% transduction efficacy; “+” means10-25% transduction efficacy; “++” means 25-50% transduction efficacy;“+++” means 50-75% transduction efficacy; and “++++” means >75%transduction efficacy.

TABLE 7 Transduction Efficacy of Exemplary Pretreatment AgentsPretreatment Agent Transduction Efficacy Alkyl disaccharidesn-Dodecyl-β-D-Maltoside (C12) ++++ n-Dodecyl-α-D-Maltoside (C12) ++++Sucrose monolaurate (C12) ++++ 6-Cyclohexylhexyl-b-D-Maltoside ++++n-Tridecyl-β-D-Maltoside (C13) +++ n-Tetradecyl-β-D-Maltoside (C14) ++n-Decyl-β-D-Maltoside (C10) + n-Octyl-β-D-Maltoside (C8) (0) Alkylsulfate (ionic Alkyl) Sodium dodecyl sulfate (C12) ++++ Sodium decylsulfate (C10) + Sodium octyl sulfate (C8) (0) Sodium tetradecyl sulfate(C14) (0) Na salt of dodecyl benzenesulfonic acid + Alkyl (ether)alcohols Polidocanol ++ Polymeric surfactants Triton X-100 + PoloxamersF68, F127 (0) Tween 20, Tween 80 (0)

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

All publications cited herein are hereby incorporated by reference intheir entirety.

1-93. (canceled)
 94. A composition comprising: a transduction enhancingagent; and an oncolytic virus; wherein the transduction enhancing agenthas a structure represented by the following general formula (I) or thefollowing general formula (II):

wherein x is a positive integer; and wherein the concentration of thetransduction enhancing agent is less than 0.025 wt/% of the composition.95. (canceled)